The structure of chromatin plays a critical role in regulating eukaryotic gene expression. In eukaryotes, DNA exists as a complex with nucleosome core particles, which consist of an octamer of two dimers each of the core histone proteins H2A-H2B and H3-H4. Nucleosome particles can further interact with linker histone H1, which binds the linker DNA at the nucleosome entry-exit point. While H1 has long been known to facilitate the formation of higher order, compacted chromatin structures in vitro, until recentl the role of H1 in regulating gene expression in vivo has been poorly understood. Studies in our laboratory have revealed an important and previously unknown role for H1 in the regulation of core histone post-translational modifications (PTMs), which play a key role in the regulation of transcription. In Drosophila, H1 is involved in regulating the deposition of the silencing modification H3K9 methylation in pericentric heterochromatin, through the recruitment of the histone methyltransferase Su(var)3-9. In mammalian cells, H1 inhibits H3K4 methylation, an activating mark, by blocking access of the H3K4 methyltransferase to nucleosomes. In both cases, the role of H1 regulation appears to be locus-specific, with H1 regulating only a select number of gene loci. These studies provide a new understanding of H1 as a major player in the regulation of histone epigenetics and gene expression. However, many critical questions remain unanswered. In particular, it is not yet clear how extensively H1 is involved in regulating the numerous core histone PTMs that are known to affect gene expression. Further, the mechanism(s) that enables H1 to achieve highly selective regulation of gene expression remains unknown. This study aims to address the role of Drosophila H1 in the regulation of core histone PTMs and the ability of H1 to impart locus-specific effects on gene expression. Based on preliminary evidence that H1 is required for H3K27 methylation, a well-established mark of the Polycomb Group (PcG) silencing pathway, I propose to perform genetic and biochemical experiments to determine how H1 interacts with PcG silencing and to elucidate the mechanistic basis for H1-dependent methylation of H3K27. A screening approach, using purified chromatin analyzed by Western blotting and mass spectrometry, will be used to determine what other core histone PTMs are regulated by H1 in vivo. Finally, the mechanisms underlying locus-specific regulation of transcription by H1 will be examined through using computational analyses, biochemical assays, and recently acquired H1 chromatin occupancy data by ChIP-seq. The results of this study will provide insight into the emerging role of H1 as a critical player in core histone epigenetics, and will shed light on the ability of H1 to function a a locus-specific regulator of gene expression in vivo.